CHEMOKINES AND IMMUNE CELLS IN HIND LIMB ISCHEMIA

描述（由申请人提供）：肌肉再生和血管生成是肢体创伤和/或缺血性损伤后肢体挽救的重要组成部分。我们已经证明，敲除小鼠缺乏单核细胞趋化蛋白-1 （MCP-1）或其特异性受体，CC趋化因子受体2 (CCR2)，在缺血或毒性损伤后巨噬细胞募集和肌肉再生方面受到损害。此外，CCR2-/-小鼠在再生肌肉中表现出增加的脂肪细胞积累。用野生型（WT）骨髓（放射嵌合体）替代CCR2 -/-小鼠的骨髓（BM）可增加早期巨噬细胞募集和正常肌肉再生（即再现WT小鼠的表型）。此外，用CCR2 -/- BM替代WT小鼠的BM导致巨噬细胞募集减少和肌肉再生受损（即再现了CCR2 -/-小鼠的表型）。这表明脑梗死来源的细胞调节肌肉的愈合反应，巨噬细胞可能是脑梗死来源的细胞类型，介导CCR2 -/-小鼠受损的肌肉再生。进一步的研究显示，与WT小鼠相比，CCR2 -/-小鼠血管生成受损，同时组织血管内皮生长因子（VEGF）降低。有趣的是，在CCR2 -/-和WT小鼠中，VEGF恢复到基线水平与最大毛细血管密度的发展有关。虽然CCR2 -/-小鼠的肌肉再生受损可归因于脑源性细胞，但尚未研究脑源性细胞与宿主源性细胞在血管生成方面的影响。我们的长期目标是确定炎症的影响，包括趋化因子系统，在血管生成和骨骼肌再生。以下3个特定目标将验证一个总体假设，即脑转移源性细胞（尤其是单核/巨噬细胞）的募集和激活对血管生成至关重要，血管生成是骨骼肌损伤后再生的关键组成部分。1)确定CCR2在脑基源性细胞和非脑基性细胞中表达对损伤后骨骼肌血管生成的贡献，2)确定MCP-1/CCR2轴对血管生成的体外和体内影响，3)确定肌肉损伤前和组织修复过程中持续的选择性和完全单核细胞/巨噬细胞消融对损伤后炎症和血管生成的影响。提出的研究具有创新性，因为它们将有助于确定脑脊髓瘤来源的细胞对血管生成的贡献。这项研究的意义在于，更好地了解骨骼肌再生和血管生成的机制，可以设计出新的主要或辅助治疗方法，以改善肢体保留和组织工程。公共卫生相关性：腿部和手臂的伤口，伴随着巨大的肌肉缺陷和很高的截肢率，在创伤受害者中很常见，特别是在伊拉克/阿富汗战争中受伤的士兵中；需要新的治疗方法来替代缺失的肌肉，以降低截肢率并改善肢体功能。我们的研究研究了形成新肌肉（包括血管）所需的多种细胞之间的复杂关系。更好地了解新血管如何帮助肌肉从损伤中恢复可能会带来新的疗法，包括组织工程策略，以帮助患者从这些毁灭性的损伤中恢复。

DESCRIPTION (provided by applicant): Muscle regeneration and angiogenesis are important components of limb salvage following traumatic and/or ischemic injury to the extremities. We have demonstrated that knockout mice lacking either monocyte chemotactic protein-1 (MCP-1) or its specific receptor, the CC Chemokine Receptor 2 (CCR2), have impairments in macrophage recruitment and muscle regeneration following ischemic or toxic injury. In addition, CCR2-/- mice exhibit increased adipocyte accumulation in regenerated muscle. Bone marrow (BM) replacement of CCR2 -/- mice with wild type (WT) BM (radiation chimeras) led to increased early macrophage recruitment and normal muscle regeneration (i.e., recapitulated the phenotype of WT mice). Furthermore, replacing the BM of WT mice with CCR2 -/- BM resulted in decreased macrophage recruitment and impaired muscle regeneration (i.e., recapitulated the phenotype of CCR2 -/- mice). This suggests that BM-derived cells modulate the healing responses of muscle and that macrophages are the likely BM-derived cell type that mediates the impaired muscle regeneration in CCR2 -/- mice. Further studies revealed impaired angiogenesis in CCR2 -/- mice in conjunction with decreased tissue vascular endothelial growth factor (VEGF) compared to WT mice. Interestingly, restoration of VEGF to baseline levels was associated with the development of maximal capillary density in both CCR2 -/- and WT mice. While impaired muscle regeneration in CCR2 -/- mice is attributable to a BM-derived cell, the effects of BM-derived vs. host-derived cells have not been studied in regards to angiogenesis. Our long-term goal is to define the influence of inflammation, including the chemokine system, in angiogenesis and skeletal muscle regeneration. The following 3 specific aims will test the overall hypothesis that the recruitment and activation of BM-derived cells, especially monocytes/macrophages, are essential for angiogenesis, a critical component in skeletal muscle regeneration after injury. 1) Determine the contribution of CCR2 expression in BM-derived vs. non-BM- derived cells on angiogenesis in skeletal muscle after injury, 2) Determine the ex vivo and in vivo influence of the MCP-1/CCR2 axis on angiogenesis and 3) Define the effects of selective and complete monocyte/macrophage ablation prior to muscle injury and sustained throughout the time course of tissue repair on inflammation and angiogenesis after injury. The proposed studies are innovative because they will help define the contribution of BM-derived cells to angiogenesis. The significance of this research is that a better understanding of the mechanisms of skeletal muscle regeneration and angiogenesis could lead to the design of novel primary or adjuvant treatments for improved limb salvage and tissue engineering. PUBLICE HEALTH RELEVANCE: Leg and arm wounds, with large muscle defects and high amputation rates, are common in trauma victims and especially in injured soldiers from the Iraqi/Afghanistan war; new treatments to replace the missing muscle are needed to decrease amputation rates and improve limb function. Our research studies the complex relationships between the multiple cells that are needed to make new muscle, including blood vessels. A better understanding of how new blood vessels help muscle recover from injury could lead to new therapies, including tissue engineering strategies, to help patients recover from these devastating injuries.